Background and Hypothesis

Cancer cells release circulating tumor DNA (ctDNA) into the bloodstream, which has the potential to be clinically relevant as a marker of tumor clonality and a marker of clinical response. This "liquid biopsy" may provide a non-invasive approach to cancer care diagnosis and monitoring. We have optimized a PCR assay for rapid and inexpensive selection and detection of a priori sequenced targets, serving as clonal and subclonal markers of cancer evolution. Our hypothesis is that this assay will allow us to detect circulating free DNA and ctDNA while modeling tumor clonality and measuring clinical response in patients undergoing treatment for metastatic prostate cancer.


From a given panel of somatic point mutations previously identified via unbiased sequencing of matched tumor and normal samples, multiplex PCR primer mixes were generated using the IonTorrent AmpliSeq Design tool, creating locus-specific primers flanking each point mutation by approximately 60bp on each side. To the 5’ end of each forward primer, a 7-base degenerate unique molecular identifier (UMI) was synthesized, to which an additional 32-base sequence complementary to the forward Illumina Nextera adaptor was further added. To the 3’ end of the reverse primer, a 34-base sequence complementary to the reverse Illumina Nextera adaptor was added. Forward primers for each patient-specific set were pooled and hybridized to aliquots of ctDNA extracted from patient plasma. The tagged product was amplified in exponential PCR with the reverse primer pool and a full-length Nextera i7 Indexing primer. Following purification and size selection, the tagged and partial-adaptor-ligated library was amplified in additional exponential cycles of PCR with full length Nextera i5 and i7 indexing primers. Libraries were then quantified and sequenced via MiSeq.

Results and Conclusions

We have previously shown that for a series of seven patients, primers designed against clonal and subclonal mutations successfully amplified their genomic targets in 269 out of 280 amplicons, averaging 10 amplicons per library and 100,000x target coverage per amplicon. Following duplicate reduction, approximately 1,000 unique molecules were sequenced. Spike-in experiments were utilized to establish the lower limit of reliable detection. This assay is currently being applied to patients with metastatic prostate cancer who had blood drawn across multiple timepoints. We are currently analyzing these samples to model tumor clonality and treatment response across multiple patients.

Relevance and Importance

We have developed a robust, patient-specific assay for detection of ctDNA and monitoring patients over time. This assay has the potential to provide a patient-specific and non-invasive approach for monitoring treatment and remission of metastatic prostate cancer.

Citation Format: Nicholas T. Terrigino, S. Thomas Hennigan, Shana Trostel, Scott C. Wilkinson, Huihui Ye, Adam G. Sowalsky. Development of an allele-specific assay for detecting circulating tumor DNA in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 736.